PROJECT SUMMARY/ ABSTRACT Ryanodine receptors (RyR2) regulate intracellular Ca2+ release from the sarcoplasmic reticulum (SR), and are important determinants of cardiac contractility. We have recently demonstrated that the enzyme Ca2+/calmodulin-dependent protein kinase (CaMKII) phosphorylates serine 2814 (S2814) on RyR2, which increases SR Ca2+ release and serves as an important regulatory mechanism to enhance cardiac contractility at faster heart rates. On the other hand, upregulated CaMKII activity has been identified as an important signaling defect in congestive heart failure (CHF). Chronic CaMKII phosphorylation of RyR2 has been proposed as a principal cause of enhanced SR Ca2+ leak, which is an important determinant of contractile dysfunction in CHF. The long-term goal of this project is to define the cellular/molecular mechanisms by which CaMKII regulates RyR2-mediated SR Ca2+ release and cardiac contractility in normal and failing hearts, by studying knockin mice in which the CaMKII phosphorylation site serine 2814 (S2814) on RyR2 is either inactivated (S2814A) or constitutively activated (S2814D). Our hypothesis is that in normal hearts, CaMKII phosphorylates S2814 on RyR2 to increase SR Ca2+ release and cardiac contractility, whereas in failing hearts chronic CaMKII phosphorylation of RyR2-S2814 enhances diastolic leak of Ca2+ from the SR, which interferes with SR Ca2+ loading and causes depressed contractility. The specific aims are to: 1) Define the effects of CaMKII phosphorylation of RyR2 on cardiac contractility; 2) Determine whether chronic CaMKII phosphorylation of RyR2-S2814 is sufficient to induce heart failure; 3) Evaluate whether inhibition of CaMKII phosphorylation of RyR2 is therapeutic in heart failure. We propose to conduct multidisciplinary studies ranging from in vivo studies in genetically-altered mice, Ca2+ imaging studies in isolated cardiomyocytes, and biochemical and single channel measurements of RyR2 Ca2+ release channels. It is anticipated that these studies will advance our understanding of CaMKII-dependent mechanisms underlying the regulation of cardiac contractility in both normal and failing hearts. The animal models developed for this project may be particularly useful for the development of new drugs for the treatment of congestive heart failure, the leading cause of death in the United States.